The miR-15/107 miRNA group are paralogous microRNAs (miRNAs), with overlapping gene regulatory impact, that are downregulated in Alzheimer's disease (AD) brains. Our underlying hypothesis is that miR- 15/107 gene augmentation (up-regulation) will help to reduce brain cellular expression of BACE1 and tau kinases (PKA and CDK5-regulating proteins). We have strong preliminary evidence in support of this hypothesis. The overall goal of the present research proposal is to obtain information necessary to develop future miRNA-based brain therapies and to increase our knowledge about miRNAs' role in AD. Therapeutic strategies must involve either stimulating endogenous miRNA production, or, alternatively, a gene therapy approach with exogenously introduced RNA or viral vectors. The key challenges, before developing brain therapies related to miR-15/107 genes, are 1> to understand upstream stimuli that alter miR-15/107 expression in human brains, and 2> define downstream genes regulated by these miRNAs, including an assessment of the potential for manipulating the off-target effects of miR-15/107-related miRNAs. We thus propose novel experiments in human brain and in primary rat brain-derived cultured cells. Specific Aims for this research project: Specific Aim #1: Define the specific AD-relevant signaling pathways that modulate miR-15/107 gene expression. Aim 1A: Perform hypothesis-based studies in cultured rat primary brain cells anchored in our preliminary results finding that glucose levels and discrete neuroinflammatory stimuli specifically alter miR-15/107 gene expression which may suggest strategies for miRNA-based therapies. Aim 1B: Perform comprehensive miR-15/107 expression profiling in human brain to understand how parameters linked with AD risk correlate with altered miR-15/107 gene expression because miRNA neurochemical studies need to be anchored in human brain data. Specific Aim #2: Define the exact downstream mRNA targets regulated by miR-15/107 genes in primary cultured rat brain cells. Preliminary data indicate that miR-15/107 genes may be a key mechanistic link between metabolic changes and increased tau phosphorylation in human brains. Using methods that were developed and optimized in our laboratory, we will perform comprehensive analyses of miR-15/107 miRNA targets in primary cultured rat brain cells. Specific Aim #3: Manipulate and minimize off-target effects of miRNAs using RNA sequence modifications in primary cultured brain cells. Cultured neurons will be transduced with lentivirus vectors harboring altered sequences related to miR-15/107 genes. We have already generated miRNA expressing lentivirus and transduced primary cultured neurons to help optimize the potential impact of miRNA-based therapies in the brain. The overall goal is to produce reagents with optimal on-target and minimal off-target impact, in order to maximize the potential value of small RNA- based therapeutics.